In continuing studies on the architecture of pyruvate dehydrogenase complexes, we have addressed another important unresolved question, which is to determine whether the gap between the central E2 core and the outer enzyme shell is maintained by virtue of protein-protein interactions in the outer shell or by stiffness of the linker region connecting the core to the peripheral subunit binding domain. Using a combination of circular dichroism, analytical ultracentrifugation and solution NMR studies we have obtained evidence that the peptide corresponding to the linker region has an extended conformation with a persistence length of 75-89 Angstroms, consistent with the observed size of the gap. Cryo electron tomography of individual complexes with varying occupancies of enzymes in the outer shell confirmed unequivocally that the annular between the core and the outer shell was maintained even at very low E1 or E3 occupancies. These studies demonstrate unambiguously that it is the linker, rather than interactions between the outer shell enzymes, that are responsible for holding the subunits above the core. We conclude that the inner linker region of PDH enzymes are critical structural elements, serving to maintain the annular gap required for coupling the decarboxylation of pyruvate in the outer shell to the synthesis of acetyl CoA in the core. In related studies, and in collaboration with Dr. Subramaniam and colleagues, we have extended these methods to analyze the 3D structures of two other protein complexes in the nano size range: HIV-1 and Doxil nanoparticles. The work on HIV-1 has led to the first report of the structure of trimeric gp120 on the surface of infectious virions. Similarly, the structural work on nanoparticles has shown that electron tomography can be a powerful method for providing quality control on the physical characteristics of complex nanomedicine formulations such as Doxil, and useful for setting standards for evaluating quality of nanoparticle formulations.